Targeting the EGFR-SGLT1 Interaction for Cancer Therapy

ABSTRACT

A compound can destabilize a binding interaction between an epidermal growth factor receptor (EGFR) and a sodium/glucose co-transporter 1 (SGLT 1). In one embodiment, the compound is a peptide derived from the interacting domain of EGFR. In another embodiment, the peptide is administered to a patient to treat cancer.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.61/821,028 filed on May 8, 2013, which is herein incorporated byreference in its entirety.

GOVERNMENT SPONSORSHIP American Cancer Society (RSG-09-206-01)Department of Defense Prostate Cancer Research Program (W91ZSQ8334N607)BACKGROUND

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinasethat is over-active/over-expressed in the majority of cancers ofepithelial origin (Hynes N E, et al., ERBB receptors and cancer: thecomplexity of targeted inhibitors, Nature Reviews Cancer, 5(5):341-354(2005)). Inhibition of the tyrosine kinase activity of EGFR has been theprinciple strategy of EGFR based cancer therapies. However, targetingEGFR by small molecule inhibitors of receptor tyrosine kinase has notproduced satisfactory therapeutic efficacy. The general response ratesare between 10-20% across a variety of human malignancies (Weiss J.,First line erlotinib for NSCLC patients not selected by EGFR mutation:keep carrying the TORCH or time to let the flame die? Transl. LungCancer Res., 1(3):219-223 (2012); Cohen S J, et al., Phase II andpharmacodynamic study of the farnesyltransferase inhibitor R115777 asinitial therapy in patients with metastatic pancreatic adenocarcinoma,J. Clinical Oncology, 21(7):1301-1306 (2003); Dancey J E, et al.,Targeting epidermal growth factor receptor—are we missing the mark?,Lancet 362(9377):62-64 (2003)). In other words, there is majorpopulation of cancer patients that do not respond to EGFR tyrosinekinase inhibitors. For example, although EGFR is over-expressed in morethan 80% of late stage prostate cancers and negatively correlates withprognosis, prostate cancer is resistant to EGFR inhibitors (Herres E.,et al., Expression of the epidermal growth factor receptor family inprostate carcinoma before and during androgen-independence, British J.Cancer, 90(2):449-454 (2004); Pu Y S, et al., Epidermal growth factorreceptor inhibitor (PD168393) potentiates cytotoxic effects ofpaclitaxel against androgen-independent prostate cancer cells,Biochemical Pharmacology, 71(6):751-760 (2006); Sherwood E R, et al.,Epidermal growth factor-related peptides and the epidermal growth factorreceptor in normal and malignant prostate, World J. Urology,13(5):290-296 (1995); Zellweger T., et al., Expression patterns ofpotential therapeutic targets in prostate cancer, International J.Cancer, 113(4):619-628 (2005); Canil C M, et al., Randomized phase IIstudy of two doses of gefitinib in hormone-refractory prostate cancer: atrial of the National Cancer Institute of Canada-Clinical Trials Group,J. Clinical Oncology, 23(3):455-460 (2005); Gross M, et al., A phase IItrial of docetaxel and erlotinib as first-line therapy for elderlypatients with androgen-independent prostate cancer, BMC Cancer 7:142(2007)).

Evidence indicates that EGFR possesses tyrosine kinase independentfunctions. For example, EGFR knockout animals die soon after birth(Threadgill D W, et al., Targeted disruption of mouse EGF receptor:effect of genetic background on mutant phenotype, Science,269(5221):230-234 (1995). However, mice with severely compromised EGFRtyrosine kinase activity are completely viable and display only someepithelial defects (Luetteke N C, et al., The mouse waved-2 phenotyperesults from a point mutation in the EGF receptor tyrosine kinase, Genes& Development, 8(4):399-413 (1994)). As another example, both a wildtype and a kinase-dead EGFR enhanced the survival of EGFR negative 32Dhematopoietic cells (Ewald J A, et al., Ligand- and kinaseactivity-independent cell survival mediated by the epidermal growthfactor receptor expressed in 32D cells, Experimental Cell Research282(2):121-131 (2003). It has also been discovered that EGFRparticipates in the maintenance of basal intracellular glucose level ofcancer cells by interacting with and stabilizing the sodium-glucoseco-transporter 1 (SGLT1), independent of EGFR tyrosine kinase activity(Weihua Z, et al., Survival of cancer cells is maintained by EGFRindependent of its kinase activity, Cancer Cell, 13(5):385-393 (2008)).

SGLT1 is an active glucose transporter that relies on extracellularsodium concentration to transport glucose into cells independent ofglucose concentration (Wright E M, et al., Biology of human sodiumglucose transporters, Physiological Reviews, 91(2):733-794 (2011). SGLT1plays a critical role in glucose absorption and retention in the body(Castaneda-Sceppa C, et al., Sodium-dependent glucose transporterprotein as a potential therapeutic target for improving glycemic controlin diabetes, Nutrition Reviews, 69(12):720-729 (2011)). One of thehallmarks of cancer is that cancer cells exhibit altered energymetabolism, i.e. cancer cells consume a substantially higher amount ofnutrients and energy substrates than their normal counterparts (HanahanD, et al., Hallmarks of cancer: the next generation, Cell,144(5):646-674 (2011). This enhanced energy consumption demands a highrate of nutrients uptake, which is achieved by over-expression of plasmamembrane transporters (Ganapathy V, et al., Nutrient transporters incancer: relevance to Warburg hypothesis and beyond, Pharmacology &Therapeutics 121(1):29-40 (2009). Studies have found that SGLT1 isover-expressed in various types of cancers including ovarian carcinoma,oral squamous cell carcinoma, colorectal cancer, pancreatic cancer, andprostate cancer (Lai B, et al., Overexpression of SGLT1 is correlatedwith tumor development and poor prognosis of ovarian carcinoma, Archivesof Gynecology and Obstetrics, 285(5):1455-1461 (2012); Hanabata Y, etal., Coexpression of SGLT1 and EGFR is associated with tumordifferentiation in oral squamous cell carcinoma, Odontology/the Societyof the Nippon Dental University, 100(2):156-163 (2012); Guo G F, et al.,Overexpression of SGLT1 and EGFR in colorectal cancer showing acorrelation with the prognosis, Medical Oncology 28 Suppl 1:S197-203(2011); Casneuf V F, et al., Expression of SGLT1, Bcl-2 and p53 inprimary pancreatic cancer related to survival, Cancer Investigation26(8):852-859 (2008); Blessing A, et al., Sodium/Glucose Co-transporter1 Expression Increases in Human Diseased Prostate, J. Cancer Sci. Ther.4(9):306-312 (2012). As an example, late stage prostate cancers expresselevated levels of EGFR and uptake a high amount of glucose (Herres E.,et al., Expression of the epidermal growth factor receptor family inprostate carcinoma before and during androgen-independence, British J.Cancer, 90(2):449-454 (2004); Pu Y S, et al., Epidermal growth factorreceptor inhibitor (PD168393) potentiates cytotoxic effects ofpaclitaxel against androgen-independent prostate cancer cells,Biochemical Pharmacology, 71(6):751-760 (2006); Sherwood E R, et al.,Epidermal growth factor-related peptides and the epidermal growth factorreceptor in normal and malignant prostate, World J. Urology,13(5):290-296 (1995); Lee S T, et al., PET in prostate and bladdertumors, Seminars in Nuclear Medicine 42(4):231-246 (2012); Oyama N, etal., The increased accumulation of [18F]fluorodeoxyglucose in untreatedprostate cancer, Japanese J. Clinical Oncology, 29(12):623-629 (1999)).A better understanding of the functional relationship between EGFR andSGLT1 may lead to identification of novel therapeutic targets for cancertherapy.

Thus, there is need in the art for methods and compositions that canadequately treat cancer cells resistant to EGFR tyrosine kinaseinhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description,will be better understood when read in conjunction with the appendeddrawings. For the purpose of illustration only, there is shown in thedrawings certain embodiments. It's understood, however, that theinventive concepts disclosed herein are not limited to the precisearrangements and instrumentalities shown in the figures.

FIG. 1A illustrates a schematic diagram of different constructs of humanEGFR, in accordance with embodiments. FIG. 1B illustrates animmunoprecipitation coupled Western blot analysis of interactionsbetween mutated EGFRs and SGLT1, in accordance with embodiments.

FIG. 2A illustrates a Western blot analysis of expression levels ofSGTL1 in HEK293 cells co-transfected with the WT-EGFR, the KD-EGFR andthe ΔAutophos-EGFR, in accordance with embodiments. FIG. 2B illustratesa densitometric quantification of bands in the Western blot of FIG. 2A,in accordance with embodiments. FIG. 2C illustrates a Western blotanalysis of the effect of a proteasome inhibitor MG132 on thedown-regulation of SGLT1 by ΔAutophos-EGFR, in accordance withembodiments. FIG. 2D illustrates a densitometric quantification of bandsin the Western blot of FIG. 2C, in accordance with embodiments.

FIG. 3A illustrates an immunoprecipitation coupled Western blot analysisof interactions between EGFR-HA and SGLT1-Flag in HEK293 cells treatedwith EGF or AEE788, in accordance with embodiments. FIG. 3B illustratesimmunoprecipitation coupled Western blot analysis of interactionsbetween endogenous EGFR and SGLT1 in PC3 cells treated with EGF orAEE788, in accordance with embodiments.

FIG. 4A illustrates co-localization of SGLT1 and EGFR in prostate cancertissues from a prostate cancer tissue array, in accordance withembodiments. FIG. 4B illustrates a Western blot analysis of expressionsof endogenous EGFR and SGLT1 in PC3 and LNCaP cells, in accordance withembodiments. FIG. 4C illustrates an MTT assay showing the effect ofinhibition of SGLT1 on the growth inhibitory effect of Gefitinib andErlotinib on PC3 cells, in accordance with embodiments. FIG. 4Dillustrates an MTT assay showing the effect of inhibition of SGLT1 onthe growth inhibitory effect of Gefitinib and Erlotinib on LNCaP cells,in accordance with embodiments.

FIG. 5A illustrates disruption of EGFR-SGLT1 interaction by smallmolecules of peptides to destabilize both proteins, in accordance withembodiments. FIG. 5B illustrates that treatment of PC3 cells with anEGFR-SGLT1 disrupting peptide (MTG-01) significantly down-regulates EGFRand SGLT1 proteins, in accordance with embodiments.

FIG. 6 illustrates the viability of cancerous PC3, Du145, HCT116, andMDA-MB-231 cells and treatment of non-cancerous HEK293 cells whentreated with MTG-01, in accordance with embodiments.

SUMMARY

A summary of certain embodiments disclosed herein is set forth below.It's understood that this section is presented merely to provide thereader with a brief summary of certain embodiments and that thesedescriptions are not intended to limit this application's scope. Indeed,this disclosure may encompass a variety of embodiments that may not beset forth herein.

The present application discloses methods and compositions for treatingcancer. In one embodiment, a compound destabilizes a binding interactionbetween an epidermal growth factor receptor (EGFR) and a sodium/glucoseco-transporter 1 (SGLT 1). In another embodiment, the compound is aPhlorizin-like compound or a peptide derived from the interacting domainof EGFR.

In an embodiment, a compound that destabilizes the binding interactionbetween EGFR and SGLT 1 is administered to a subject intratumorally,intravenously, or systemically. The compound may be a Phlorizin-likecompound or a peptide derived from the interacting domain of EGFR. Inyet another embodiment, the compound may be administered in conjunctionwith a tyrosine-kinase inhibitor.

DETAILED DESCRIPTION

Before explaining at least one embodiment in detail, it should beunderstood that the inventive concepts set forth herein are not limitedin their application to the construction details or componentarrangements set forth in the following description or illustrated inthe drawings. It should also be understood that the phraseology andterminology employed herein are merely for descriptive purposes andshould not be considered limiting.

It should further be understood that any one of the described featuresmay be used separately or in combination with other features. Otherinvented systems, methods, features, and advantages will be or becomeapparent to one with skill in the art upon examining the drawings andthe detailed description herein. It's intended that all such additionalsystems, methods, features, and advantages be protected by theaccompanying claims.

All references cited in this application are incorporated by referencein their entirety.

Over-expression of epidermal growth factor receptor (EGFR) is associatedwith poor prognosis in malignant tumors. Sodium/glucose co-transporter 1(SGLT1) is an active glucose transporter that is over-expressed incancers including prostate cancer. It has been found that EGFR interactswith and stabilizes SGLT1 in cancer cells.

As explained in extensive detail below, the following embodiments havebeen identified in this application:

-   -   the critical micro-domain of EGFR that is required for its        sufficient interaction with and mutual stabilization with SGLT1;    -   the effects of activation/inactivation of EGFR on EGFR-SGLT1        interaction;    -   the measured expression of EGFR and SGLT1 in prostate cancer        tissues and cell lines;    -   the effect of inhibition of SGLT1 on the sensitivity of prostate        cancer cells to EGFR tyrosine inhibitors;    -   the amino acid sequence of a synthesized peptide, ESD-01;    -   the effect of ESD-01 on the stability of EGFR and SGLT1 proteins        in cancer cells; and    -   the effects of ESD-01 on survivability of non-cancer cells        (HEK293) and several types of cancer cells cultured in vitro.    -   the ESD-01 peptide made of either L-amino acids or D-amino acids        are equally effective in killing cancer cells in vitro.

In one embodiment, the autophosphorylation region (978-1210 amino acids)of EGFR is required for its sufficient interaction with SGLT1. Thisinteraction is independent of EGFR's tyrosine kinase activity. Mostimportantly, in another embodiment, the EGFR-SGLT1 interaction isirresponsive to EGFR tyrosine kinase modulators (EGF and tyrosine kinaseinhibitors). In yet another embodiment, EGFR and SGLT1 co-localize inprostate cancer tissues. In still another embodiment, inhibition ofSGLT1 by a SGLT1 inhibitor (Phlorizin) sensitizes prostate cancer cells(PC3 and LNCaP) to EGFR inhibitors (Gefitnib and Erlotinib). In afurther embodiment, ESD-01 (SEQID: 001) destabilizes the EGFR-SGLT1interaction. All of this data suggests that EGFR in cancer cells canexit in two types of statuses—a tyrosine kinase modulator responsivestatus and an irresponsive status. Therefore, in an embodiment, SGLT1 isa protein involved in EGFR's functions that are irresponsive to EGFRtyrosine kinase inhibitors, and the EGFR-SGLT1 interaction may be anovel target for prostate cancer therapy.

EGFR Protein Region Required for Interaction with SGLT1

In one embodiment, to determine the EGFR protein region required for itsinteraction with SGLT1, a flag tagged SGLT1²³ and HA tagged EGFRs havinga variety of mutations may be created (Blessing A, et al.,Sodium/Glucose Co-transporter 1 Expression Increases in Human DiseasedProstate, J. Cancer Sci. Ther. 4(9):306-312 (2012)). FIG. 1A, by way ofexample only, illustrates a schematic diagram of human EGFR constructsthat may be used to determine the EGFR protein region required for itsinteraction with SGLT1. For example, in some embodiments, the constructsmay include: wild type EGFR (“WT”); kinase dead EGFR (R817M) (“KD”)(SEQID: 012); transmembrane domain deletion (645-670aa) (“ATM”) (SEQID:013); extracellular domain deletion (1-644aa) (“ΔExtra”) (SEQID: 014);intracellular domain deletion (671-1210aa) (“ΔIntra”) (SEQID: 015);tyrosine kinase domain deletion (670-977aa) (“ΔTK”) (SEQID: 016); orautophosphorylation domain deletion (978-1210aa) (“ΔAutophos”) (SEQID:017).

In an embodiment, the flagged SGLT1 and above identified HA tagged EGFRscan be transiently co-transfected into HEK293 cells. In anotherembodiment, SGLT1 can be immunoprecipitated using anti-flag antibodies.In yet another embodiment, Western blot analyses may be performed for HAtagged EGFRs. FIG. 1B, by way of example only, illustrates a Westernblot analysis of interactions between mutated EGFRs and SGLT1, whereIP=immunoprecipitation, IB=Immunoblot, and Input=expression levels ofindicated exogenous proteins in HEK293 whole cell lysates used for theimmunoprecipitation. As shown in FIG. 1B, deletion of the entireintracellular domain or the autophosphorylation domain of EGFRsubstantially diminishes its interaction with SGLT1. In one embodiment,the autophosphorylation domain of EGFR is required for its sufficientinteraction with SGLT1.

Previously, using EGFR's extracellular domain and an intracellulardomain that does not contain the EGFR TM domain, it was discoverred thatthe extracellular domain of EGFR interacted with SGLT1 better than itsintracellular domain (Weihua Z, et al., Survival of cancer cells ismaintained by EGFR independent of its kinase activity, Cancer Cell13(5):385-393 (2008)). In an embodiment, to further characterize theEGFR-SGLT1 interaction at the plasma membrane, the TM domain may beincluded into the constructs of truncated EGFRs. In one embodiment, theTM containing intracellular domain of EGFR, especially theautophosphorylation domain, interacts more strongly with SGLT1 than itsextracelluar domain. The discrepancy between this embodiment and thedata shown in the previous report is very likely due to the lack of TMdomain in the intracellular domain construct used in the previous study.

EGFR's Autophosphorylation Domain Required to PreventProteasome-Mediated SGLT1 Degradation

In an embodiment, to determine whether the autophosphorylation domain ofEGFR is required to sustain the stability of SGLT1, the expression levelof SGLT1 co-transfected with the WT-EGFR, the KD-EGFR, and theΔAutoPhos-EGFR into HEK293 cells may be measured. For example, FIG. 2A,by way of example only, illustrates a Western blot analysis ofexpression levels of SGTL1 in HEK293 cells co-transfected with theWT-EGFR, the KD-EGFR and the ΔAutophos-EGFR. The same amounts of DNAplasmids of SGLT1 and EGFRs may be used in each group of treatments.Furthermore, control cells can be transfected with the same amount ofDNA of the empty vector. Actin may be used as loading control. FIG. 2B,by way of example only, illustrates a densitometric quantification ofbands in the Western blot of FIG. 2A. Asterisk marks indicatestatistical significance between the linked representative groups fromtriplicate experiments.

As shown in FIGS. 2A-2B, in one embodiment, the level of SGLT1 in theWT-EGFR and the KD-EGFR transfected cells is much higher than that inthe control vector or ΔAutoPhos-EGFR transfected cells. In anotherembodiment, the autophosphorylation domain of EGFR can maintain theexpression level of SGLT1. In still another embodiment, the level ofSGLT1 in the ΔAutoPhos-EGFR transfected cells is significantly lowerthan that of the control cells. In yet another embodiment, the loss ofSGLT1 interaction with EGFR can promote down-regulation of SGLT1.

In an embodiment, to determine whether proteasome is involved in loss ofinteraction with EGFR induced down-regulation of SGLT1, SGLT1 andΔAutoPhos-EGFR co-transfected HEK293 cells can be treated with aproteasome inhibitor, such as but not limited to MG231. For example,FIG. 2C illustrates a Western blot analysis of the effect of aproteasome inhibitor MG132 on the down-regulation of SGLT1 byΔAutophos-EGFR. Actin may be used as a loading control. FIG. 2D, by wayof example only, illustrates a densitometric quantification of bands inthe Western blot of FIG. 2C. Asterisk marks indicate statisticalsignificance between the linked representative groups from triplicateexperiments.

As shown in FIGS. 2C-2D, by way of example only, in one embodiment MG231can inhibit the down-regulation of SGLT1 in ΔAutoPhos-EGFR transfectedcells. In another embodiment, the proteasome machinery is involved inthe loss of interaction with EGFR induced SGLT1 down-regulation. Inother words, proteasomes may cause the ultimate degradation of SGLT1when the EGFR-SLGT1 interaction is compromised. In yet anotherembodiment, the proteasome machinery may be a potential therapeutictarget alone or in combination with the EGFR-SLGT1 interaction.

In one embodiment, deleting the SGLT1 interacting domain in EGFRpromotes the down-regulation of SGTL1 via the proteasome machinery. Inanother embodiment, this disruption of the EGFR-SGLT1 interaction inEGFR positive cancer cells can lead to down-regulation of SGLT1.Furthermore, previous data shows that knocking down SGLT1 by shRNAresults in autophagic cell death of prostate cancer cells (Weihua Z, etal., Survival of cancer cells is maintained by EGFR independent of itskinase activity, Cancer Cell 13(5):385-393 (2008). In an embodiment, theEGFR-SGLT1 interaction can be a significant target to improve EGFR basedtherapy for cancer.

EGFR-SGLT1 Interaction is Irresponsive to EGFR Tyrosine KinaseInhibitors

In an embodiment, the effects of EGFR tyrosine kinase inhibitors onEGFR's interaction with SGLT1 can be determined. In an embodiment,WT-EGFR and SGLT1 co-transfected HEK293 cells can be treated with eitherEGF or an EGFR tyrosine kinase inhibitor, AEE788. SGLT1 may beimmunoprecipitated, and the levels of EGFR that wereco-immunoprecipitated with SGLT1 can be measured. For example, FIG. 3A,by way of example only, illustrates an immunoprecipitation coupledWestern blot analysis of interactions between EGFR-HA and SGLT1-Flag inHEK293 cells treated with EGF or AEE788, where EGFR=total EGFR,pEGFR=phosphorylated EGFR, IP=immunoprecipitation, IB=immunoblot, andInput=expression levels of indicated exogenous proteins in HEK293 wholecell lysates used for the immunoprecipitation. As shown in FIG. 3A, inone embodiment, neither EGF nor AEE788 significantly affects theEGFR-SGLT1 interaction. In yet another embodiment, the EGFR thatco-precipitates with SGLT1 is not phosphorylated.

In one embodiment, to determine the effects of EGF and AEE788 onendogenous EGFR-SGLT1 interaction and the phosphorylation status ofendogenous EGFR that interacts with SGLT1, endogenous SGLT1 of PC3 cellstreated with EGF or AEE788 may be immunoprecipitated. In anotherembodiment, the phosphorylation status of the EGFR co-precipitated withSGLT1 can be measured. By way of example only, FIG. 3B illustrates animmunoprecipitation coupled Western blot analysis of interactionsbetween endogenous EGFR and SGLT1 in PC3 cells treated with EGF orAEE788. In an embodiment, neither EGF nor AEE788 affect the EGFR-SGLT1interaction. In yet another embodiment, the endogenous SGLT1 interactingEGFR is not phosphorylated. In still another embodiment, the EGFR-SGLT1interaction is irresponsive to modulators of EGFR's tyrosine kinaseactivity. In yet a further embodiment, EGFR can be targeted for cancertherapy at its non-kinase functionality, including but not limited toits interaction with SGLT1.

It has been well documented that, upon phosphorylation of tyrosineswithin the autophosphorylation domain of EGFR, the autophosphorylationdomain serves as a major docking site for recruitment ofadaptor/effector proteins that transactivate downstream signaling(Bazley L A, et al., The epidermal growth factor receptor family,Endocrine-Related Cancer, 12 Suppl 1:S17-27 (2005)). Based on theresults of FIG. 3A, in an embodiment, the EGFR-SGLT1 interaction isindependent of EGFR activation/inactivation. The autophosphorylationdomain of EGFR functions as a protein-protein interacting domainindependent of EGFR's tyrosine kinase activity. In one embodiment, EGFRpossesses pro-survival functions independent of its tyrosine kinaseactivity. For example, EGFR can exist in two types of statuses—atyrosine kinase responsive status and a tyrosine kinase irresponsivestatus. Upon activation by EGFR's ligands, the autophosphorylationdomain of the kinase responsive EGFR can be phosphorylated and canrecruit effectors to trigger downstream signals. Alternatively, thekinase irresponsive EGFR may constantly interact with proteinsregardless of the presence of EGFR ligands, and regardless of activationor inactivation of its tyrosine kinase. In yet another embodiment, SGLT1may be one of such proteins that bind to and keep EGFRs in their kinaseirresponsive status. In yet another embodiment, the non-phosphorylatedautophosphorylation domain of EGFR may be used as a tool for identifyingtherapeutic targets.

Inhibition of SGLT1 by SGLT1 Inhibitor Sensitizes Prostate Cancer Cellsto EGFR Inhibitors

In an embodiment, to determine the clinical relevance of the EGFR-SGLT1interaction, immunofluorescent co-staining of EGFR and SGLT1 on a tissuemicroarray of prostate cancers (n=44) may be performed. For example,FIG. 4A, by way of example only, illustrates the results of threerepresentative prostate cancer tissues from a prostate cancer tissuearray. In an embodiment, SGLT1 (green) and EGFR (red) may beco-localized in the prostate tissue (e.g., orange or yellow, asindicated by arrows). In another embodiment, stromal cells may bepositive for SGLT1, but negative for EGFR (e.g., indicated by arrowheads). In yet another embodiment, in EGFR positive cancer samples(n=41), SGLT1 co-localizes with EGFR in cancer cells, but not stromalcells.

In still another embodiment, the EGFR-SGLT1 interaction may contributeto the pathogenesis of prostate cancer. For example, in an embodiment,the co-localization of EGFR with SGLT1 in prostate cancer tissues canindicate that the EGFR-SGLT1 interaction is cancer relevant. Currently,at the clinic, EGFR tyrosine kinase inhibitors have not yet shownsatisfactory therapeutic effects for prostate cancer (Canil C M, et al.,Randomized phase II study of two doses of gefitinib inhormone-refractory prostate cancer: a trial of the National CancerInstitute of Canada-Clinical Trials Group, J. Clinical Oncology,23(3):455-460 (2005); Gross M, et al., A phase II trial of docetaxel anderlotinib as first-line therapy for elderly patients withandrogen-independent prostate cancer, BMC Cancer 7:142 (2007)). In oneembodiment, considering the fact that EGFR expression correlates withdisease progression of prostate cancer and the clinical unresponsivenessof prostate cancers to EGFR tyrosine kinase inhibitors, EGFR maycontribute to the disease progression of prostate cancer independent ofits tyrosine kinase activity. Additionally, previous findings concludethat (1) prostate cancer tissues have increased expression of SGLT1,²³(2) a loss of EGFR protein but not its tyrosine kinase activitysensitized prostate cancer cells to chemotherapeutic agent,²⁹ and (3) aloss of EGFR induced autophagic cell death was mediated bydown-regulation of SGLT1 protein (Blessing A, et al., Sodium/GlucoseCo-transporter 1 Expression Increases in Human Diseased Prostate, J.Cancer Sci. Ther. 4(9):306-312 (2012); Xu S, et al., Loss of EGFRinduced autophagy sensitizes hormone refractory prostate cancer cells toadriamycin, The Prostate. 17:1216-1224 (2011); Weihua Z, et al.,Survival of cancer cells is maintained by EGFR independent of its kinaseactivity, Cancer Cell, 13(5):385-393 (2008)). In yet another embodiment,EGFR can promote prostate cancer progression via stabilizing SGLT1 tosustain the high demand of glucose of late stage cancer cells. Thisembodiment is not only supported by all the embodiments describedherein, but is also supported by past data that over-expression of SGLT1prevented renal epithelial cells and intestinal epithelial cells fromapoptosis (Ikari A, et al., Sodium-dependent glucose transporter reducesperoxynitrite and cell injury caused by cisplatin in renal tubularepithelial cells, Biochimica et Biophysica Acta 1717(2):109-117 (2005);Yu L C, et al., SGLT-1-mediated glucose uptake protects human intestinalepithelial cells against Giardia duodenalis-induced apoptosis,International journal for parasitology 38(8-9):923-934 (2008)).

It is well known that an increase in glucose levels can activate EGFR,that SGLT1 is over-expressed in prostate cancer tissues, and thatprostate cancer is resistant to EGFR inhibitors. (Han L, et al., Highglucose promotes pancreatic cancer cell proliferation via the inductionof EGF expression and transactivation of EGFR, PloS One 6(11):e27074(2011); Blessing A, et al., Sodium/Glucose Co-transporter 1 ExpressionIncreases in Human Diseased Prostate, J. Cancer Sci. Ther. 4(9):306-312(2012); Canil C M, et al., Randomized phase II study of two doses ofgefitinib in hormone-refractory prostate cancer: a trial of the NationalCancer Institute of Canada-Clinical Trials Group, J. Clinical Oncology,23(3):455-460 (2005); Gross M, et al., A phase II trial of docetaxel anderlotinib as first-line therapy for elderly patients withandrogen-independent prostate cancer, BMC Cancer 7:142 (2007)). In anembodiment, SGLT1 and EGFR can synergistically promote prostate cancergrowth. In one embodiment, to test whether inhibition of SGLT1 cansensitize prostate cancer cells to EGFR inhibitors, prostate cancer celllines (e.g., PC3 and LNCaP (both positive for EGFR and SGLT1)), may betreated with EGFR tyrosine kinase inhibitors (e.g., Gefitnib, Erlotinib,Icontinib, Mubritinib, Vandertanib, Lapatinib, Pelitinib, Canertinib,Neratinib, Afatinib and Dacomitinib.) in the presence/absence of anSGLT1 inhibitor (e.g., phlorizin, and phlorizin derivatives, such asCanagliflozin and Dapagliflozin) (Ehrenkranz J R, et al., Phlorizin: areview, Diabetes/Metabolism Research and Reviews 21(1):31-38 (2005)). Inone embodiment, the growth inhibitory effects of the treatments may bedetermined. For example, in an embodiment, cells can be treated with theSGLT1 inhibitor phlorizin (50 μM) with/without EGFR inhibitors(Gefitinib, 10 μM; Erlotinib, 10 μM) for 48 hs before being subjected toMTT assay. The OD value of control cells can be artificially set as 1.All experiments may be repeated for at least 3 times. Asterisk marksindicate statistical significances between linked groups.

FIG. 4B, by way of example only, illustrates a Western blot analysis ofthe expressions of endogenous EGFR and SGLT1 in PC3 and LNCaP cells. Inone embodiment, breast and colon cancer cells express EGFR and SGLT1.FIG. 4C shows an MTT assay of the effect of inhibition of SGLT1 on thegrowth inhibitory effect of Gefitinib and Erlotinib on PC3 cells. FIG.4D shows an MTT assay of the effect of inhibition of SGLT1 on the growthinhibitory effect of Gefitinib and Erlotinib on LNCaP cells. Based onthese results, in one embodiment, co-inhibition of SGLT1 and EGFRfunctions can more effectively inhibit cancer cell growth. In stillanother embodiment, Phlorizin can significantly sensitize prostatecancer cells to the growth inhibitory effects of Gefitnib and Erlotinib.In an embodiment, Phlorizin or Phlorizin-like compounds can beadministered to a patient, intravenously, intratumorally, orsystemically, to treat cancer. In still another embodiment, Phlorizin orPhlorizin-like compounds can be administered to a patient in conjunctionwith tyrosine-kinase inhibitors to treat cancer.

In another embodiment, a peptide can destabilize EGFR and SGLT1 proteinsof cancer cells. FIG. 5A, by way of example only, illustrates anembodiment of a peptide, called ESD-01 (SEQID: 001), which candestabilize EGFR and SGLT1 proteins of cancer cells. In one embodiment,the ESD-01 peptide can be selected from the SGLT1 interacting domain ofEGFR. In another embodiment, ESD-01 comprises amino acids 1049-1062 of ahuman EGFR protein (GenBank: AAH94761.1). In still another embodiment,the effects of ESD-01 on the stability of EGFR and SGLT1 in culturedcells (e.g., PC3, MDA-MB-231, and HCT116 cells) can be determined byWestern blot analysis. FIG. 5B, by way of example only, shows thatESD-01 treatment (20004 for 6 hours) significantly down-regulates EGFRand SGLT1 levels in PC3 cells, which are inhibited by the proteasomeinhibitor MG132. In an embodiment, inhibition of SGLT1 and EGFRdown-regulation by ESD-01 by MG132 suggests that MTG-01 destabilizesEGFR and SGLT1 via proteolysis. In still another embodiment, the effectsof ESD-01 on the survivability of cultured cancer cells (e.g., PC3,MDA-MB-231, and HCT116 cells) can be determined by trypan blue uptakeassay. FIG. 6, by way of example only, shows that ESD-01 treatment(10004 for 24 hours) significantly reduces the survivability of avariety of cancer cells including prostate cancer PC3 and Du145 cells,breast cancer MDA-MB-231 cells, and colon cancer HCT116 cells. In yetanother embodiment, ESD-01 treatment cannot reduce the survivability ofnon-cancerous HEK293 cells.

It is well known that replacement of amino acids in a protein/peptidewith different amino acids having similar chemical properties cangenerate proteins/peptides with identical functions. In an embodiment,ESD-01 (SEQID: 001) can be substituted with amino acids having similarchemical properties or any other mutations that will destabilize EGFRand SGLT1 proteins of cancer cells. For example, in one embodiment, theamino acids can be either L-form or D-form chiral isomers. In anotherembodiment, Serine (S) in ESD-01 can be substituted with Threonine (T),Tyrosine (Y), or any other non-natural hydroxyl containing amino acid(e.g., SEQID: 002). In yet another embodiment, Threonine (T) in ESD-01can be substituted with Serine (S), Tyrosine (Y), or any othernon-natural hydroxyl containing amino acid (e.g., SEQID: 003). In stillanother embodiment, Lysine (K) in ESD-01 can be substituted withTheronine (T) (e.g., SEQID: 004). In an embodiment, Glutamine (Q) can inESD-01 can be substituted with Histidine (H) (e.g., SEQID: 005). Inanother embodiment, Threonine (T) at position 10 of ESD-01 can besubstituted with Alanine (A) (e.g., SEQID: 006). In yet anotherembodiment, where the Serine (S) at position 12 of ESD-01 can besubstituted with Leucine (L) (e.g., SEQID: 007). In still anotherembodiment, the polar positive charged amino acids in ESD-01, Lysine (K)and Histidine (H), can be any other natural and non-natural positivelycharged amino acid (e.g., SEQID: 008). In one embodiment, the polaramino acids in ESD-01, Glutamine (Q) and Cysteine (C), can be any othernatural and non-natural polar amino acid (e.g., SEQID: 009). In anotherembodiment, the Cysteine (C) in ESD-01 can be any other natural andnon-natural thiol side chain (—SH) containing amino acid (e.g., SEQID:010). In still another embodiment, the nonpolar amino acids in ESD-01,Leucine (L), Valine (V), and Tryptophan (W), can be any other naturaland non-natural nonpolar amino acids (e.g., SEQID: 011).

It's understood that the above description is intended to beillustrative, and not restrictive. The material has been presented toenable any person skilled in the art to make and use the inventiveconcepts described herein, and is provided in the context of particularembodiments, variations of which will be readily apparent to thoseskilled in the art (e.g., some of the disclosed embodiments may be usedin combination with each other). Many other embodiments will be apparentto those of skill in the art upon reviewing the above description. Thescope of the invention therefore should be determined with reference tothe appended claims, along with the full scope of equivalents to whichsuch claims are entitled. In the appended claims, the terms “including”and “in which” are used as the plain-English equivalents of therespective terms “comprising” and “wherein.”

Examples Cells and Reagents

HEK293 cell line, prostate cancer cell line PC3, LNCaP, Du145,MDA-MB-231, and HCT116 cells were originally obtained from the AmericanType of Culture Collection (ATCC) and maintained in DMEM supplementedwith 10% fetal bovine serum and 1% Penicillin/Streptomycin under 5% CO₂at 37° C. Mouse anti-Flag-tag antibody (F1804), proteasome inhibitorMG231, and phlorizin dihydrate were obtained from Sigma-Aldrich (St.Louis, Mo.). AEE788, Gefitinib, and Erlotinib were obtained fromSelleckchem (Houston, Tex.). Antibody against pEGFR (Y1173) (cat. no.2434L) was obtained from Cell Signaling (Danvers, Mass.). Monoclonalantibody against C225 was obtained from Dr. Lee Elis (M.D. AndersonCancer Center). Rabbit anti-actin (cat. no. sc-7210), rabbit anti-HA-tagantibody (sc-805), secondary antibodies against rabbit and mouse labeledwith horseradish peroxidase, and protein A/G conjugated agarose beads(cat. no. sc-2003) were obtained from Santa Cruz Biotechnology (SantaCruz, Calif.). MTT kit (cat. no. 30-1010K) was obtained from ATCC. Theplasmid expressing flag tagged human SGLT1 and the rabbitanti-human-SGLT1 polyclonal antibodies for immunohistochemical analysis(SGLT1-IHC) and Western blotting analysis (SGLT1-WB) have beenpreviously described (Blessing A, et al., Sodium/Glucose Co-transporter1 Expression Increases in Human Diseased Prostate, J. Cancer Sci. Ther.4(9):306-312 (2012)).

Plasmid Constructions.

Human wild type EGFR was cloned into a pcDNA3.1 vector (Clontech, CA),which was used as a parental vector to generate all the other EGFRconstructs. The pRK5 expression plasmid (Clontech, CA) with a c-terminalHA tag was used for the constructions of all HA tagged EGFRs. Thefull-length human EGFR was amplified with a forward primer EGFR-F(ATTCTCGAGCGGGGAGCAGCGATG) and a reverse primer EGFR-R(CCTAAGCTTTGCTCCAATAAATTCACTG). DNA fragments were digested by Xho I andHind III and cloned into the corresponding sites of the pRK5 vector.Primers for cloning the EGFR with extracellular domain deletion (ΔExtra,1-644aa) are ΔExtra-F: ATTCTCGAGATGTCC ATCGCCACTGGGATG and ΔExtra-R:CCTAAGCTTTGCTCCAATAAATTCACTGC; primers for intracellular deletion(Alntro, 671-1210 aa) are ΔIntra-F: TATCTCGAGATGCGACCCTCCGGGACGGC andΔIntra-R: CCTAAGCTTCC TTCGCATGAAGAGGCC; primers for autophosphorylationdomain deletion (ΔAutophos, 978-1210aa) are ΔAutophospho-F:ATTCTCGAGATGTCCATCGCCACTGGGATG and ΔAutophospho-R:CCTAAGCTTGTAGCGCTGGGGGTCTCGG; primers for intracellular domain deletion(645-1210aa) are ΔIntra-F: TATCTCGAGATGCGACCCTCCGGGACGGC and ΔIntra-R:CCTAAGCTTCCTTCGCATGAAGAGGC. The kinase dead mutant of EGFR (KD-EGFR,R817M), transmembrane domain deletion (ATM, 645-670aa), and tyrosinekinase domain deletion (ATK, 670-977aa) plasmids were constructed frompRK5-WT-EGFR-HA by site-directed mutagenesis using the QuikChangeLightning Site-Directed Mutagenesis Kit (Agilent, CA) according to themanufacturer's protocol. The primers were: KD-EGFR-F:GCACCGCGACCTGGCAGCC ATGAACGTACTGGTGAAAACACC and KD-EGFR-R:GGTGTTTTCACCAGTACGTTCATGGCTGCCA GGTCGCGGTGC; ΔTM-F:CGAGACCCCCAGCGCTACCGGACTCCCCTCCTGAGC and ΔTM-R:CGAGACCCCCAGCGCTACCGGACTCCCCTCCTGAGC; ΔTK-F: CGCTGCGGAGGCTGCTGCAGTACCTTGTCATTCAGGGGG and ΔTK-R: CCCCCTGAATGCAAGGTACTGCAGCAGCCTCCGCAGCG. Allof the constructs yielded fusion proteins with a C-terminal HA tag. Allplasmids were confirmed by sequencing.

Transient Transfection and Immunoprecipitation.

HEK293 cells were transfected with plasmids expressing flagged SGLT1alone or with indicated HA tagged EGFR constructs. After 24 hours oftransfection, cells were washed in 1× phosphate buffered solution andlysed with RIPA buffer (50 mM Tris-HCl, pH 8.0, with 150 mM sodiumchloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1%sodium dodecyl sulfate), supplemented with a protease inhibitorscocktail for 6 hours on a shaker at 4° C. The cell lysates were thencentrifuged for 2 minutes at 12000×rpm. Supernatant were then incubatedwith sepharose protein A/G beads conjugated with anti-flag or anti-SGLT1antibody for overnight at 4° C. Samples were then centrifuged and washedwith RIPA buffer three times before boiled in Laemmle buffer (Biorad,CA) and subjected to Western blot analysis. To determine the role ofEGFR's tyrosine kinase in EGFR-SGLT1 interaction, HEK293 cells weretransfected with SGLT1 and wild type EGFR. After 18h, cells were starvedin serum free medium for 6h before EGF treatment (10 ng/ml), or EGF plusAEE788 (5 μM) for 30-60 min. Control cells were treated with an equalvolume of vehicle dimethyl sulfoxide (DMSO). Cell lysates were thensubjected to immunoprecipitation as described above.

Western Blot Analysis.

For Western blot (WB) analysis, cells were lysed with RIPA buffer (150mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.1% SDS, 1% TritonX-100, 1 mM EDTA, 1mM PMSF, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 20 μg/ml pepstatine, 1%sodium deoxycholate, 1 mM NaF, 1 mM Na₃VO₄, in H₂O). Proteins separatedby 8% SDS-PAGE were transferred to PVDF membrane followed by blockingwith 5% nonfat dry milk and then incubation with primary antibodies atoptimized concentrations for overnight at 4° C. The membranes werewashed with 0.1% TBS/T (1×TBS, 0.1% Tween-20) 3 times, each time for 5min before incubation with secondary antibody for 1 hour at roomtemperature. Signals were visualized by enhanced chemiluminescence.

Immunofluorescent Co-Staining.

For immunofluorescent co-staining of SGLT1 and EGFR, slides of prostatecancer tissue array were deparrafinated and rehydrated before antigenswere retrieved in boiling citrate buffer for 10 min. Cooled tissueslides were then incubated in a blocking solution (5% donkey serum inPBS) for 1 hour at room temperature and then overnight at 4° C. with therabbit polyclonal antibody against SGLT1 (SGLT1-IHC) (1:200 dilution)and C225 (1:200) in PBS containing 10% donkey serum (Blessing A, et al.,Sodium/Glucose Co-transporter 1 Expression Increases in Human DiseasedProstate, J. Cancer Sci. Ther., 4(9):306-312 (2012)). After being washedthree times with PBS, tissues were incubated with a mixture of AlexaFluor 488-conjugated donkey anti-rabbit IgG and Alexa Flour594-conjugated donkey anti-mouse IgG dissolved in PBS containing 10%donkey serum for 30 minutes at room temperature. The stained sampleswere then washed three times (5 minutes per wash) with PBS at roomtemperature. Fluorescence images were captured and analyzed with aconfocal microscope (Olympus). Cell nucleus was stained by4′,6-diamidino-2-phenylindole (DAPI).

Cell Growth Assay.

Cell growth was determined by3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium Bromide (MTT)assay in 96-well plates according to the protocol provided by themanufacture. Briefly, 5000 cells suspended in 100 μL medium were seededin each well of a 96-well plate. On the second day, medium was replacedby medium containing Phlozidin (50 μM) and EGFR inhibitors (Iressa: 20μM; Erlotinib: 20 μM). After 24 or 48 hours incubation with drugs, 10 μLMTT reagents was added to each well and incubated for 4 hours. Afterremoval of the medium, the formazan precipitates in cells were dissolvedin 100 μL DMSO. Absorbance was measured by a MultiSkan plate reader(Thermo Fisher Scientific, NC) at 570 nm. Triplicates of sample in eachgroup were used. Cell viability was determined by trypan blue uptakeassay. The percentage of live cells were counted in areas (n=3 for eachsample) randomly selected under 10× magnificence of triplicates.

Statistical Analysis.

The Student's t test was used to assess the difference in growth ofcells treated with EGFR inhibitors in the presence/absence of SGLT1inhibitor. P values less than 0.05 were defined as having statisticalsignificance.

What is claimed is:
 1. A composition comprising: a compound thatdestabilizes a binding interaction between an epidermal growth factorreceptor (EGFR) and a sodium/glucose co-transporter 1 (SGLT 1).
 2. Thecomposition of claim 1, wherein the compound is at least one of apeptide, and a peptide analog.
 3. The composition of claim 2, whereinthe peptide is derived from the SGLT 1 interacting domain of EGFR. 4.The composition of claim 2, wherein the peptide comprises the amino acidsequence LVWKQSCSSTSSTH (SEQID: 001).
 5. The composition of claim 2,wherein the peptide comprises a compound substantially similar to atleast one of SEQID: 002, SEQID: 003, SEQID: 004, SEQID: 005, SEQID: 006,SEQID: 007, SEQID: 008, SEQID: 009, SEQID: 0010, and SEQID:
 0011. 6. Thecomposition of claim 1, wherein the compound is at least one of aPhlorizin SGLT 1 inhibitor, and a Phlorizin-like SGLT 1 inhibitor.
 7. Amethod of treating cancer cells in a subject comprising: administeringto the subject a compound that destabilizes a binding interactionbetween an epidermal growth factor receptor (EGFR) and a sodium/glucoseco-transporter 1 (SGLT 1).
 8. The method of claim 7, wherein thecompound is at least one of a peptide, and a peptide analog.
 9. Themethod of claim 8, wherein the peptide comprises the amino acid sequenceLVWKQSCSSTSSTH (SEQID: 001).
 10. The method of claim 8, wherein thepeptide comprises at least one of SEQID: 002, SEQID: 003, SEQID: 004,SEQID: 005, SEQID: 006, SEQID: 007, SEQID: 008, SEQID: 009, SEQID: 0010,and SEQID:
 0011. 11. The method of claim 7, further comprisingadministering to the subject a tyrosine-kinase inhibitor.
 12. The methodof claim 7, further comprising administering to the subject a compoundthat targets the proteasome machinery.
 13. The method of claim 7,wherein the cancer cells are at least one of breast cancer cells,prostate cancer cells, and colon cancer cells.
 14. The method of claim8, further comprising identifying targets for cancer therapy by usingthe peptide to identify downstream survival pathways controlled by theEGFR-SGLT1 interaction.
 15. A method of treating cancer cells in asubject comprising: administering to the subject a tyrosine-kinaseinhibitor and at least one of a Phlorizin SGLT 1 inhibitor, and aPhlorozin-like SGLT 1 inhibitor.
 16. The method of claim 15, wherein theadministering destabilizes a binding interaction between an epidermalgrowth factor receptor (EGFR) and a sodium/glucose co-transporter 1(SGLT 1).
 17. The method of claim 15, further comprising administeringto the subject a tyrosine-kinase inhibitor.
 18. The method of claim 17,wherein the cancer cells are prostate cancer cells.
 19. The method ofclaim 17, wherein the tyrosine-kinase inhibitor is at least one ofGefitnib, Erlotinib, Icontinib, Mubritinib, Vandertanib, Lapatinib,Peletinib, Canetinib, Neratinib, Afatinib, and Dacomitinib.
 20. Themethod of claim 15, wherein the Phlorizin SGLT 1 inhibitor is at leastone of Canagliflozin, and Dapagliflozin.